The amount and type of dietary fat consumed is a factor in the risk for several chronic health problems in humans. Yet, our understanding of the molecular mechanisms of fat transport and assimilation and its regulation at the cellular level remains incomplete. The proposed project continues investigation focused specifically on the subcellular coordination required for peroxisomal oxidation of fatty acids. The yeast Saccharomyces cerevisiae is particularly suited as a model system, as fatty acid oxidation is restricted to peroxisomes in this organism. In addition, yeast is a simple eukaryote for which the entire genome has been sequenced and characterized and a plethora of genetic tools is available. Published and preliminary results have revealed a requirement for the mitochondrial oxodicarboxylate transporters, Odclp and Odc2p, and two uncharacterized transporters, Ymclp and Ymc2p, for growth on fatty acids as sole carbon source. An array of biochemical, NMR, cell biological and genetic techniques will be used to clarify the role of these transporters in the assimilation of fatty acids and coordination of peroxisomal and mitochondrial metabolism in yeast. The specific aims to be pursued are: (1) To characterize the expression and subcellular localization of the transporters in normal and mutant cells, (2) to examine carbon source metabolism in strains carrying disrupted transporter genes and (3) to directly assay specific biomolecules related to possible in vivo functions of the transporters. It is anticipated that these studies will provide insight into the cellular function(s) of these transporters in yeast as well as the mechanisms by which eukaryotes coordinate peroxisomal and mitochondrial metabolism of fatty acids.